Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

From 1959 -1964, the Prince Edward County, VA School Board closed down its public schools to circumvent the 1954 Brown v. Board of Education Supreme Court ruling declaring separate public schools for Black and White students "inherently unequal" and the 1955 Brown II ruling to desegregate public schools with "all deliberate speed." For five years, more than 1700 African American children received no public education in the county, as White children attended a newly-constructed and private Prince Edward Academy. While some students left Prince Edward to reside with relatives, others were placed with families by the American Friends Service Committee. However, the majority of Black children remained in the county without formalized public instruction. This study investigated the perceived impact of The Closing on adult self-directed learning, lifelong learning, occupational choices and success within a family with sixteen of its twenty-one children forced from school. Via audio-/video-taped interviews, three participants reflected upon their "lived experiences" during and since The Closing. Transcribed data were coded and analyzed based upon the major and underlying research questions guiding the study. Nine major conclusions were drawn from its findings: (a) The Closing perceivably impacted immediate educational goals of participants differently, (b) The Closing perceivably impacted specific and general long-range educational goals, (c) Participants have pursued educational goals via supportive spouses/family members and adult self-directed/lifelong learning measures, (d) Following the re-opening of schools, all respondents graduated high school, and two later enrolled in academic learning centers, (e) Self-directed learning has played an essential role in the lives of all participants, (f) All participants considered themselves life-long learners, (g) The Closing perceivably impacted the career plans of one participant, (h) Respondents acquired manufacturing and/or labor positions and were successfully employed throughout their adult lives, (i) Literacy assistance from family members, self-directed learning, on-the-job training and formalized coursework were perceived as having had a positive bearing on occupational success. The implications of this study suggested resiliency, family dynamics, family values, and narratological significance. Study participants, driven to live productive and successful lives, appeared to have emulated Adult Learning Theory tenets of self-directed, lifelong quests for formally-delivered and informally-acquired knowledge. Recommendations emerging from this study included investigations of School Closing survivors' motivations for adult learning, the role of faith in Closing survivors' lives, The Closing's perceived impact on the Next Generation, ancestral discourse, male birth order relationships, 1951 strikers' guilt, education vs. vocation and growth under adversity. / Ph. D.

Thesis advisor: Tim van Opijnen / Although vaccines and antibiotics have been historically successful in combating bacterial infections, limited vaccine coverage and the rise of antibiotic resistance emphasize the need to develop alternative, broadly effective, and/or targeted treatment strategies to reduce the health burden of bacterial infections. Rather than relying on therapeutics solely targeting the bacterial pathogen, such as standard antibiotics, therapies that simultaneously focus on host responses are emerging. In this thesis, we propose 'host-informed therapies' (HITs) in two categories: those that aid patients with fully functional immune systems and those that aid patients with perturbed immune processes, as promising alternative or adjunctive treatment strategies for bacterial infections. The host-pathogen interaction during infection is a highly dynamic process between diverse bacterial pathogens and hosts with varying degrees of susceptibility. Systems biology approaches have provided an understanding of host-pathogen parameters globally through the detection of putative biomarkers for diagnosis and identification of critical interactions to discover novel drug targets. However, there remains a gap in understanding bacterial pathogenesis in the context of designing novel host-informed therapies. Here, we use Streptococcus pneumoniae, the gram-positive pathogen responsible for the majority of bacterial respiratory tract infections worldwide, as a case study to: (1) Generate a genome-wide map of bacterial immune (complement) evasion targets to design novel host-informed therapies, (2) generate a dual host/pathogen transcriptome map to identify signatures of infection outcome, and (3) validate signatures of bacterial antibiotic tolerance in a mouse lung infection model. Overall, this work exemplifies how systems biology methods can elucidate the intricacies of bacterial pathogenesis but, more importantly, aid in the target identification, validation, and design of antibacterial host-informed therapies. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The overall goal of this work is to create an open-source digital twin of the wire arc directed energy deposition process using robot operating system 2 for interpass temperature regulation of a maraging steel alloy. This framework takes a novel approach to regulating the interpass temperatures by using in-situational infrared camera data and a closed loop feedback control that is enabled by robot operating system 2. This is the first implementation of robot operating system 2 for wire arc directed energy deposition and this framework outlines a sensor and machine agnostic approach for creating a digital twin of this additive manufacturing process. In-situ control of the welding process is conducted on a maraging steel alloy demonstrating interpass temperature regulation leads to improved as-built surface roughness and more consistent as-built hardness. An evaluation of three distinct weld modes: Pulsed MIG, CMT MIX, and CMT Universal and two primary process parameters: travel speed and wire feed speed was conducted to identify suitable process windows for welding the maraging alloy. Single track welds for each parameter and weld mode combination were produced and evaluated against current weld bead metrics in the literature. Non destructive profilometry and destructive characterization were performed on the single track welds to evaluate geometric features like wetting angle, dilution percentage, and cross sectional area. In addition, the role of material feed rate on heat input and the cross sectional area was examined in relation to the as-built hardness. The robot operating system 2 digital twin provides a visualization environment to monitor and record real time data from a variety of sensors including robot position, weld data, and thermal camera images. Point cloud data is visualized, in real time, to provide insight to the captured weld meta data. Capturing in-situ data from the wire arc directed energy deposition process is critical to establishing an improved understanding of the process for parameter optimization, tool path planning, with both required to build repeatable, quality components. This work presents an open-source method to capture multi-modal data into a shared environment for improved data capture, data sharing, data synchronization, and data visualization. This digital twin provides users enhanced process control capabilities and greater flexibility by utilizing the robot operating system 2 as a middleware to provide interoperability between sensors and machines.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC family are characterized by the presence of two helix-turn-helix (HTH) motifs, which bind to the promoter DNA and activate transcription. ExsA uses its HTH containing C-terminal domain (CTD) to regulate gene expression from 10 different promoters. The N-terminal domain (NTD) of ExsA mediates dimerization and regulation of ExsA-activity. While most AraC-type activators are regulated by a small molecule ligands, ExsA is regulated by another protein, ExsD. As part of a four-protein signaling cascade, ExsD interacts directly with ExsA to prevent transcription of T3SS-associated genes under non-inducing conditions prior to host cell contact. The entire regulatory cascade includes of two additional proteins, ExsC and ExsE. ExsA, ExsC, ExsD, and ExsE follow a partner-switching mechanism to link expression of the secretion system with host cell contact. Our laboratory is working to understand this unique signaling mechanism by determining the molecular basis for the regulation of this important virulence factor. Previous studies in the laboratory have solved the structures of ExsE, ExsC and ExsD, and shed light on how these proteins interact and compete for overlapping binding sites. However, it is still unclear as to how the ExsA and ExsD interact and thus how regulation is mediated at the molecular level. In the presented study, we sought to map the molecular interface between ExsA and ExsD. First, the crystal structure of ExsA-NTD is presented wherein the dimerization interface of the protein was identified. Two of the well-studied AraC-type proteins, AraC and ToxT crystal structures have been solved by others in the presence of their respective ligands. Residues that were involved in ligand binding in AraC and ToxT were aligned with the residues in ExsA and analyzed for interaction with ExsD. However, this canonical binding pocket appeared to be not involved in the interaction between ExsA and ExsD. Structure directed site-specific mutagenesis was carried out to construct many different variants of ExsD and ExsA. Thus constructed variants were purified and analyzed in a functional assay. Using this approach, we were able to identify regions on ExsD and ExsA that are crucial for the interaction and for the regulation of ExsA-dependent transcription. It turns out that backbone interactions between the amino-terminal residues of ExsD and the beta-barrel region of the ExsA-NTD are pivotal. This result explains how ExsA and ExsC compete for ExsD binding, since both target the same regions on ExsD. / PHD / Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for causing severe infections in immunocompromised individuals. Acute Pseudomonas aeruginosa infections are characterized by immediate adverse effects. An initial acute infection may become chronic, leading to long-term morbidity and mortality in affected individuals. During the initial stages of infection P. aeruginosa uses the type three secretion system, a syringe-like structure, to puncture the host cell and inject potent toxins. The activation of the genes required for forming this structure is tightly controlled by an activator protein, ExsA. When P.aeruginosa is not invading a host, ExsA is inhibited by another protein called ExsD, to prevent the needless production of the secretion apparatus. The presented work explores the mechanism of how ExsD achieves this inhibition of ExsA. This information is of potential biomedical interest because a clear understanding of the molecular basis for the interaction could inform the development of a small-molecule mimic of ExsD to be used in therapy. In Chapter 2 we report the structure of the domain of ExsA that is known to bind ExsD. Also, in this chapter and more so in Chapter 3, we performed a detailed analysis of potential interacting regions and ultimately succeeded in identifying key interacting regions in both ExsA and ExsD.

The RNA world hypothesis posits that at some stage in the development of life, RNA functioned as both an informational polymer and a catalyst for important reactions. However, many questions remain as to how RNA molecules might have evolved into living organisms. This thesis uses computer simulations to model processes thought to be important to the development of an RNA world. First, a model is discussed which describes non-enzymatic polymerization of single-stranded RNA from different kinds of activated nucleotides, a necessary first step towards an RNA world. It was found that a system undergoing polymerization of RNA from 5′-activated triphosphates or imidazolides behaves differently from an equilibrium system undergoing reversible polymerization reactions from 2′,3′-cyclic monophosphates, for example. In the 5′-triphosphate case, the system is not in equilibrium but rather in a state of circular reaction flux that must be maintained by an external source of phosphates. This model is then adapted to investigate non-enzymatic template-directed replication of RNA strands. It is found that this process fulfills all the necessary requirements to function as a metabolism which maintains a difference between the outside non-living environment and the internal environment of the cell. Finally, byproducts arising from the template copying mechanism in this model are discussed, including the development of highly regular sequence patterns in the strand population due to selection for the ability to form duplexes with neighbouring strands. Altogether, this thesis illustrates new implications, potential pitfalls, and possibilities of the RNA world hypothesis for the origin of life. In particular, it emphasizes the fundamental link between the processes of replication and metabolism, both of which must have been crucial to the functioning of the earliest protocells. This link has been largely overlooked in scientific literature on the topic to date. / Thesis / Master of Science (MSc) / For millennia, humanity has told stories about the origin of life. Since the 1960s, scientists have hypothesized that RNA is a key player in this origin story. RNA can both hold information and catalyze chemical reactions, meaning only one molecule is needed for both these crucial functions. However, many questions remain about how this would work in practice. This project used computer simulations to model steps along the path from RNA to living organisms. First, a model was developed for the formation of single-stranded RNA from building block molecules. The model was then expanded to include copying of existing RNA strands, and it was found that this process constitutes a metabolism. Finally, it was discovered that over time the copying process produces simple patterns in the sequence of building blocks that make up the RNA strands. Altogether, these findings emphasize the link between replication and metabolism in early cells.

The Technological Immersion Learning Theory (TILT) was developed through a classic grounded theory study in the seminal tradition of Glaser and Strauss (1967) and Glaser (1978, 1992, 1998, 2001, 2007). The purpose of the study was to investigate an exemplary case of self-determined technology enthusiasts in the hopes of generating a substantive grounded theory that conceptualizes their experiences and concerns. Twelve unstructured interviews of amateur radio enthusiasts from the eastern United States provided the initial / primary data for this study. Experimenting and self-teaching in technological activities was highlighted as the main concern of the participants. The basic social process (BSP) of technological immersion learning (TIL) emerged as a theoretical construct and core variable that illuminates the experiences of individuals immersed in a community of practice, where hands-on engagement with technology is a primary activity. Adventuring, Affirmation, Doing Technology, Experimenting, Overcoming Challenge, Self-teaching, and Social Networking were properties of technological immersion learning that interact dialectically in an amplifying causal loop, with Problem solving and Designing as active sub processes in response to unmet challenges. TIL occurs cyclically in three stages, beginning with Induction, a credentialing stage wherein the neophyte is prepared with the necessary knowledge and skill to become a novice participant in an activity. The transition from Induction into the Immersion phase is a status passage whereby the novice is absorbed into the technical culture of the group and commences autonomous active participation in hands-on experimenting. Hands-on experiences with experimenting, problem solving and social interactions provide diverse learning and affirmation for the doer and multiple sources of feedback that promote sustained engagement. The transition into the Maturation phase proceeds gradually over time, with prolonged engagement and cumulative gains in knowledge, skill, and experience. Maturation is a quasi-stable state that remains responsive to new contexts as a random-walk process, wherein trigger events can initiate new cycles of technological immersion learning in a perpetually evolving process of personal development. Engagement, Empowerment, and Self-Actualization are underlying dimensions of the TIL basic social process that provide the impetus for continued persistence and personal development. / Ph. D. / This research was undertaken to investigate the unique phenomenon of people who successfully pursue complex technological activities as a pastime, yet may possess no formal training in their field. This example of informal learning is little charted and warranted deeper study through grounded theory research. The study was conducted utilizing the classic grounded theory method that was pioneered by Glaser and Strauss in the 1960s for the specific purpose of generating theory from empirical data. The outcome of the study was the <i>Technological Immersion Learning Theory (TILT)</i>, a new theory of learning that highlights the importance of technological activities as a foundation for transdisciplinary learning. <i>TILT</i> is an organic theory of learning rigorously grounded in data to conceptualize the latent behavior patterns of individuals immersed in a community of practice devoted to hands-on experimenting and learning through technology. The technical, social, and psychological attributes of TILT illuminate complex technological environments where sustained engagement and multidimensional feedback can promote learning and personal development. As a basis of learning, technology is intertwined with virtually every aspect of human experience, transcending the artificial boundaries of academic disciplines. <i>TILT</i> offers an empirical model for the study of technology as a foundation of education relevant for the modern technological world.

京都大学 / 新制・課程博士 / 博士(情報学) / 甲第24734号 / 情博第822号 / 新制||情||138(附属図書館) / 京都大学大学院情報学研究科社会情報学専攻 / (主査)教授 緒方 広明, 教授 伊藤 孝行, 准教授 馬 強 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

Thesis advisor: Abhishek Chatterjee / Genetic code expansion (GCE) is an enabling technology whereby noncanonical amino acids (ncAAs) can be site-specifically incorporated into proteins of interest, allowing for vast applications and an improved understanding of structure-function relationships in biology. GCE stands out as a versatile platform due to the use of a variety of engineered aminoacyl-tRNA synthetase (aaRS)/transfer RNA (tRNA) pairs, and it has endowed proteins with over 200 distinct ncAAs in both prokaryotic and eukaryotic systems. My dissertation outlines endeavors aimed at broadening the chemical diversity of α-amino side chains and substrates beyond α-amino acids in both prokaryotic and eukaryotic organisms through the utilization of GCE technology. This was achieved by creating universal GCE platforms called altered translational machinery (ATM) strains, which eliminate the limitations of orthogonality for the evolution of aaRS/tRNA pairs. This expansion enables the use of the same aaRS/tRNA pair for ncAA incorporation functionalities into multiple domains of life. Moreover, the diversity of ncAAs that can be genetically encoded in eukaryotic cells was enhanced by evolving the E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNA pair using a yeast-based selection system. This advancement facilitated the incorporation of novel ncAAs into proteins within mammalian cells. Additionally, I worked toward developing a platform for introducing monomers into the genetic code beyond α-amino acids. This involved developing an aaRS evolution platform that doesn't rely on translation as a selectable readout. Finally, I worked towards the creation of polyester-polyamide oligomers with sequence control as a step towards the goal of generating sequence-defined biopolymers with new-to-nature backbone chemistries. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.